Use of PTC124 for nonsense suppression therapy targeting BMP4 nonsense variants in vitro and the bmp4st72 allele in zebrafish.

Nonsense suppression therapy (NST) utilizes compounds such as PTC124 (Ataluren) to induce translational read-through of stop variants by promoting the insertion of near cognate, aminoacyl tRNAs that yield functional proteins. We used NST with PTC124 to determine if we could successfully rescue nonsense variants in human Bone Morphogenetic Protein 4 (BMP4) in vitro and in a zebrafish bmp4 allele with a stop variant in vivo. We transfected 293T/17 cells with wildtype or mutant human BMP4 cDNA containing p.Arg198* and p.Glu213* and exposed cells to 0-20 μM PTC124. Treatment with 20 μM PTC124 produced a small, non-significant increase in BMP4 when targeting the p.Arg198* allele, but not the p.Glu213* allele, as measured with an In-cell ELISA assay. We then examined the ability of PTC124 to rescue the ventral tail fin defects associated with homozygosity for the p.Glu209* allele of bmp4 (bmp4st72/st72) in Danio rerio. We in-crossed bmp4st72/+ heterozygous fish and found a statistically significant increase in homozygous larvae without tail fin and ventroposterior defects, consistent with phenotypic rescue, after treatment of dechorionated larvae with 0.5 μM PTC124. We conclude that treatment with PTC124 can rescue bmp4 nonsense variants, but that the degree of rescue may depend on sequence specific factors and the amount of RNA transcript available for rescue. Our work also confirms that zebrafish show promise as a useful animal model for assessing the efficacy of PTC124 treatment on nonsense variants.